丝素蛋白—羟基磷灰石骨材料的纳米结构设计及骨诱导性能调控研究
发布时间：2018-11-07 11:28
【摘要】：由于创伤、感染以及发育异常等原因导致的大面积骨缺损的修复在临床治疗上仍面临巨大的挑战。目前常用的自体和异体移植治疗方法都存在不同程度的问题。组织工程的出现为骨缺损修复的治疗提供了新的选择方法。构建生物特性与正常骨相似的组织工程骨是目前骨组织工程研究的热点。羟基磷灰石的组成与人体骨的无机成分相似。尤其是纳米级羟基磷灰石,因具有良好的生物相容性、骨传导性和骨再生性,被广泛应用于骨组织工程领域。但目前的纳米级羟基磷灰石存在尺寸均一性和水分散性较差的问题,限制了其在骨组织工程中的应用。从仿生矿化的角度出发,利用纳米技术,制备水分散性优良的纳米羟基磷灰石,构建新型实用的骨组织修复材料,有望推动骨组织工程的发展。丝素蛋白由于其独特的机械性能、生物相容性和缓慢的降解性,受到越来越多的关注,成为骨组织工程的基质材料。本文首先以尺寸均一的丝素蛋白纳米颗粒为模板仿生矿化制备纳米级羟基磷灰石,通过调控工艺参数,获得尺寸均一,能在水中稳定分散、具有核壳结构的羟基磷灰石/丝素蛋白纳米颗粒,并研究发现了丝素蛋白包覆层对稳定纳米颗粒的关键作用。随后,将此纳米颗粒直接分散在丝素蛋白溶液中形成均一的丝素蛋白与羟基磷灰石混合物,经过冷冻干燥制备丝素蛋白/羟基磷灰石复合支架。与已有报道的丝素蛋白/羟基磷灰石复合支架相比,本研究所制备的复合支架不仅具有更高的羟基磷灰石含量,同时实现了羟基磷灰石纳米颗粒在纳米尺度的均匀分布,以及力学强度的显著提高。体外细胞实验结果表明,在不添加成骨生长因子的情况下,复合支架中羟基磷灰石的含量和分布影响骨髓间充质干细胞的生长和成骨分化能力。纳米颗粒含量高且分布均匀的丝素蛋白复合支架能够提供更适宜的环境,显著提高间充质干细胞的生长和成骨分化能力。与此同时,利用上述具有核壳结构的羟基磷灰石/丝素蛋白纳米颗粒作为载体,成功加载骨生长因子骨形态蛋白2(BMP-2),并实现了因子的可控释放。BMP-2加载率高达99.6%,可稳定释放21天以上,没有出现“突释”现象。细胞实验结果表明同原有体系相比,现有的控释体系能够更好地促进间充质干细胞的生长和向成骨细胞的分化。随后,将BMP-2控释体系同丝素蛋白/羟基磷灰石复合支架制备技术结合,构建能够可控释放BMP-2,且羟基磷灰石均匀分布的复合支架体系,以构建适合骨修复的微环境。同不含BMP-2的复合支架相比,上述体系不仅获得了更好的成骨分化效果,同时还通过调控BMP-2的释放行为实现了干细胞向成骨细胞分化能力的优化。动物实验结果进一步表明,同原有加载BMP-2的丝素蛋白基支架相比,该复合支架显示出更好地诱导骨再生的能力。总之,本文以尺寸均一的丝素蛋白纳米颗粒为模板,调控羟基磷灰石纳米颗粒的生成,不仅优化了丝素蛋白/羟基磷灰石骨材料的设计,同时实现了更为有效控释BMP-2的载体的制备以及同丝素蛋白/羟基磷灰石骨材料的复合,最终构建出适合骨再生的微环境。更重要的是,基于对上述不同因素的调控,研究了不同关键因素影响干细胞分化以及骨修复的作用,为新型骨材料的设计提供了理论参考。
[Abstract]:The repair of large-area bone defects due to trauma, infection, and developmental abnormalities still faces great challenges in clinical treatment. There are different degrees of problems in the methods of autograft and allograft treatment. The appearance of tissue engineering provides a new method for the treatment of bone defect repair. The construction of tissue engineering bone with similar biological characteristics and normal bone is a hot spot in the research of bone tissue engineering at present. The composition of the hydroxyapatite is similar to that of the human bone. in particular, that nano-level hydroxyapatite is widely used in the field of bone tissue engineering because of good biocompatibility, bone conductivity and bone regeneration. However, the present nano-grade hydroxyapatite has the problems of poor size and poor water dispersibility, and the application of the nano-hydroxyapatite in the bone tissue engineering is limited. Starting from the angle of biomimetic mineralization, nano-hydroxyapatite with excellent water dispersibility is prepared by using nano-technology, and a novel practical bone tissue repair material is constructed, which is expected to promote the development of bone tissue engineering. The silk fibroin is a matrix material for bone tissue engineering due to its unique mechanical properties, biocompatibility and slow degradation. The preparation method comprises the following steps of: firstly, performing bionic mineralization on the silk fibroin nano-particles with uniform size as a template to prepare the nano-scale hydroxyapatite, and obtaining the hydroxyapatite/ silk fibroin nano-particles with uniform size, stable dispersion in water, The key role of the coating of silk fibroin in stabilizing nanoparticles was also studied. then, the nano particles are directly dispersed in the silk fibroin solution to form a uniform silk fibroin and a hydroxyapatite mixture, and the fibroin/ hydroxyapatite composite stent is prepared by freeze drying. Compared with the existing reported silk fibroin/ hydroxyapatite composite stent, the composite stent prepared by the research institute not only has higher hydroxyapatite content, but also realizes the uniform distribution of the hydroxyapatite nano-particles in the nano-scale and the remarkable improvement of the mechanical strength. The results of in vitro cell experiment show that in the absence of bone growth factor, the content and distribution of the hydroxyapatite in the composite scaffold can affect the growth of the mesenchymal stem cells and the ability of bone differentiation. The silk fibroin composite stent with high nanometer particle content and uniform distribution can provide a more suitable environment, and can obviously improve the growth and the bone differentiation capacity of the mesenchymal stem cells. At the same time, the bone morphogenetic protein 2 (BMP-2) was loaded successfully by using the hydroxyapatite/ silk fibroin nanoparticles with the core shell structure as a carrier, and the controllable release of the factor was realized. The loading rate of BMP-2 is up to 99.6%, and can be stably released for more than 21 days without any 鈥渂urst鈥，
本文编号：2316222
[Abstract]:The repair of large-area bone defects due to trauma, infection, and developmental abnormalities still faces great challenges in clinical treatment. There are different degrees of problems in the methods of autograft and allograft treatment. The appearance of tissue engineering provides a new method for the treatment of bone defect repair. The construction of tissue engineering bone with similar biological characteristics and normal bone is a hot spot in the research of bone tissue engineering at present. The composition of the hydroxyapatite is similar to that of the human bone. in particular, that nano-level hydroxyapatite is widely used in the field of bone tissue engineering because of good biocompatibility, bone conductivity and bone regeneration. However, the present nano-grade hydroxyapatite has the problems of poor size and poor water dispersibility, and the application of the nano-hydroxyapatite in the bone tissue engineering is limited. Starting from the angle of biomimetic mineralization, nano-hydroxyapatite with excellent water dispersibility is prepared by using nano-technology, and a novel practical bone tissue repair material is constructed, which is expected to promote the development of bone tissue engineering. The silk fibroin is a matrix material for bone tissue engineering due to its unique mechanical properties, biocompatibility and slow degradation. The preparation method comprises the following steps of: firstly, performing bionic mineralization on the silk fibroin nano-particles with uniform size as a template to prepare the nano-scale hydroxyapatite, and obtaining the hydroxyapatite/ silk fibroin nano-particles with uniform size, stable dispersion in water, The key role of the coating of silk fibroin in stabilizing nanoparticles was also studied. then, the nano particles are directly dispersed in the silk fibroin solution to form a uniform silk fibroin and a hydroxyapatite mixture, and the fibroin/ hydroxyapatite composite stent is prepared by freeze drying. Compared with the existing reported silk fibroin/ hydroxyapatite composite stent, the composite stent prepared by the research institute not only has higher hydroxyapatite content, but also realizes the uniform distribution of the hydroxyapatite nano-particles in the nano-scale and the remarkable improvement of the mechanical strength. The results of in vitro cell experiment show that in the absence of bone growth factor, the content and distribution of the hydroxyapatite in the composite scaffold can affect the growth of the mesenchymal stem cells and the ability of bone differentiation. The silk fibroin composite stent with high nanometer particle content and uniform distribution can provide a more suitable environment, and can obviously improve the growth and the bone differentiation capacity of the mesenchymal stem cells. At the same time, the bone morphogenetic protein 2 (BMP-2) was loaded successfully by using the hydroxyapatite/ silk fibroin nanoparticles with the core shell structure as a carrier, and the controllable release of the factor was realized. The loading rate of BMP-2 is up to 99.6%, and can be stably released for more than 21 days without any 鈥渂urst鈥，
本文编号：2316222
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